We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Progress continues in studies on the structure of the postsynaptic density and its remodeling by drugs used in the treatment of mood disorders, on protein complexes implicated by genomic studies of schizophrenia, and synaptic protein changes accompanying long term depression (LTD). We are testing the hypothesis that bipolar disorder arises from abnormalities in cellular plasticity cascades, leading to aberrant information processing in synapses and circuits. The mood stabilizers lithium and valproate are thought to exert their therapeutic effects via actions on systems involved in synaptic plasticity. The postsynaptic density (PSD) is an elaborate cytoskeletal and signaling complex that provides anchors for synaptic proteins close to the region of presynaptic neurotransmitter release, and therefore mediates signaling in divergent signal transduction pathways. Collaborative studies with NINDS focused on defining the composition and stoichiometry of the post-synaptic density complex. We chose thirty two signature peptides observed consistently in LC/MS/MS analyses, representing the C-and N-terminal regions from 16 prominent proteins isolated with the PSD signaling complex. Two of three synthetic genes synthesized then generated stable isotope labeled internal standard proteins using in vitro translation. We investigated multiple methods to purify these products, and determined that because they are unnatural proteins, they are inherently insoluble and not readily purified by conventional protein affinity tags or chromatographic systems without large losses of material. However, they can be used directly as quantitative internal standards as precipitated from the in vitro translation preparation. All of the synthetic gene products contain a 10-mer streptavidin labeled peptide that is conveniently quantified with respect to a high purity synthetic standard (unlabeled), with the result that this method is generally applicable to all of the planned synthetic gene products. We obtained preliminary results for SynGAP and BRAG1 relative to PSD-95, but are preparing additional labeled synthetic gene products to have sufficient material for systematic repeated measures of multiple preparations of PSDs to obtain benchmark values for use in modeling studies in coordination with electron microscopy data. The goal of a second PSD project is to understand the temporal and spatial dynamics of the PSD in the treatment of mood disorders. It is our contention that in addition to manipulating key candidate molecules these studies are critical to elucidate the mechanisms of synaptic regulation and of mood stabilizer action. We isolated hippocampal PSDs from groups of rats treated chronically with lithium, valproate, or controls. Peptide mixtures from the tryptically digested PSDs, were separated by ion exchange chromatography prior to LC/MS/MS analysis on a high resolution mass analyzer. We identified 605 proteins in the PSD of the rat hippocampus, based on concatenated data sets derived from nine sets of mass spectrometric analyses (288 runs). Of the total, 597 proteins (99%) were found in both treatment groups and control (but not necessarily in all three replicates), while 332 (55%) of the proteins were found in all of the replicates of all three groups. The functional classification of the proteins showed our PSD preparation was predominantly composed of signaling proteins (23%), cytoskeletal and cell adhesion (20%) and synaptic vesicle proteins (13%) similar to those reported in surveys of PSD proteomics literature. Seven proteins were significantly altered by both lithium and valproate treatment: metabotropic glutamate receptor 3 (Grm3), ankyrin 3 (Ank3), dynein heavy chain 1 (Dyhc1), and 14-3-3 protein isoforms T, F, E and Z. All seven proteins identified responded similarly, in direction and magnitude, to both mood stabilizers. Results suggest that chronic lithium and valproate treatments do not promote synthesis or pruning of new protein families, but instead modulate significant increases or decreases in the abundance of proteins present in the postsynaptic proteome as defined by isolation methods commonly used in this field. The ErbB4 and Dystrobrevin binding protein 1 (dysbindin) protein signaling complexes have both been implicated by genomic studies on schizophrenia and have remained difficult to analyze in brain. We are continuing a project to identify, characterize and validate complexes associated with both proteins. ErbB4 is a receptor tyrosine-protein kinase, and we have used antibodies to covalently link it to magnetic beads. Comparative LC/MS/MS proteomic studies using on-bead digestion procedures developed for this project have failed to provide the enrichment factor required for ErbB4 recovery and mass spectrometric detection. However, new peptide targeting strategies are planned using labeled proteins from synthetic genes to allow better tracking of enrichment and recovery of this complex. Although the gene for Dysbindin-1/DTNBP1was identified several years ago, the molecular functions of DTNBP1 are not fully understood. DTNBP1 has been identified as a stable component of biogenesis of lysosome-related organelles complex-1 (BLOC-1) in the brain soluble fraction. Several other binding partners of DTNBP1 have also been reported individually, such as Snapin, NF-YB, AP-3, DNA-PK, WAVE2 and Abi-1. In brains, DTNBP1 is present in both soluble cytosol and insoluble synaptic fractions (synaptosome, vesicle, and PSD). Because DTNBP1 is a soluble protein, we hypothesized that DTNBP1 interacts with unknown membrane protein complexes to be able to exist in the insoluble synaptic fractions. In the past year, we have generated a polyclonal anti-DTNBP1 antibody and focused on the DTNBP1-associated protein complexes in the insoluble fraction of brains. We found that DTNBP1 is present in large protein complexes using sucrose gradient centrifugation and DSP chemical crosslinking, necessary to preserve the integrity of the DTNBP1-associated complex. We plan to test the anti-DTNBP1 antibody for immunoprecipitation and use it to purify the membrane-bound DTNBP1 complexes from mouse brains. The components in the complexes will be identified using mass spectrometry. Targeted peptide analyses of dysbindin immunoprecipitates are planned for human lymphocytes collected in genetic studies. Long-term depression (LTD) is a lasting decrease in synaptic effectiveness that involves removal and internalization of AMPA receptors from synapses. Recent studies suggest that activation of caspase-3 via mitochondria is required for LTD and AMPA receptor internalization in hippocampal neurons. Activation of caspase-3 in LTD promotes AMPA receptor internalization instead of cell death. To address how caspase-3 induces AMPA receptor internalization in LTD, we used a N-terminal labeling method followed by mass spectrometry to identify caspase-3 substrates cleaved in LTD. The N-terminal labeling method permits selective labeling and captures emergent peptides derived from proteolytic cleavage upon LTD induced by NMDA. The captured peptides then can be identified by mass spectrometry. We have successfully adopted the N-terminal labeling method by purifying subtiligase, and have optimized the labeling condition and downstream steps toward purification and mass spectrometry. We obtained a first data set using gel-based mass spectrometry after N-terminal labeling and purification from the neurons treated by none (control), NMDA (LTD induction), and staurosporine (apoptosis induction) and are presently analyzing the data.